This invention relates to a certain class of compounds, and the pharmaceutically acceptable salts, solvates and prodrugs thereof, which inhibit Procollagen C-proteinase (xe2x80x9cPCPxe2x80x9d). They are therefore useful in the treatment of mammals having conditions alleviable by inhibition of PCP. Especially of interest is an antiscarring treatment for wounds.
Fibrotic tissues, including dermal scars, are characterised by excessive accumulation of extracellular matrix, mainly collagen type I. It is thought that inhibition of collagen deposition will reduce formation of scar tissue. Collagen is secreted as the precursor, procollagen, which is transformed into the insoluble collagen by cleavage of the C-terminal propeptide by PCP. PCP is a zinc-dependent metalloprotease which is secreted from TGF-xcex2-activated fibroblasts belonging to the subfamily of astacin-like proteases and able to cleave the C-terminal peptide of types I, II and III procollagens. Furthermore, data suggest that PCP activates lysyl oxidase, an enzyme essential for the formation of covalent cross-links which stabilise the fibrous form of collagen. Therefore, inhibition of PCP may not only reduce collagen deposition but may also make collagen more accessible for degradation.
Collagen is integral to, among other things, the proper formation of connective tissue. Thus, the over- or under-production of collagen or the production of abnormal collagen (including incorrectly processed collagen) has been linked with numerous connective tissue diseases and disorders. Mounting evidence suggests that PCP is an essential key enzyme for the proper maturation of collagen (see for example International Patent Application publication number WO 97/05865).
The present invention relates to substances capable of inhibiting PCP activity in order to regulate, modulate and/or reduce collagen formation and deposition. More specifically, the invention relates to the use of compounds and pharmaceutical compositions thereof for the treatment of various conditions relating to production of collagen.
At present more than nineteen types of collagens have been identified. These collagens, including fibrillar collagen types I, II, III are synthesized as procollagen precursor molecules which contain amino- and carboxy-terminal peptide extensions. These peptide extensions, referred to as xe2x80x9cpro-regions,xe2x80x9d are designated as N- and C- propepfides, respectively. The pro-regions are typically cleaved upon secretion of the procollagen triple helical precursor molecule from the cell to yield a mature triple helical collagen molecule. Upon cleavage, the xe2x80x9cmaturexe2x80x9d collagen molecule is capable of association, for example, into highly structured collagen fibers. See e.g., Fessler and Fessler, 1978, Annu. Rev. Biochem. 47:129-162; Bornstein and Traub, 1979, in: The Proteins (eds. Neurath, H. and Hill, R. H.), Academic Press, New York, pp. 412-632; Kivirikko et al., 1984, in: Extracellur Matrix Biochemistry (eds. Piez, K. A. and Reddi. A. H.), Elsevier Science Publishing Co., Inc., New York, pp. 83-118; Prockop and Kivirikko, 1984, N. Engl, J. Med. 311:376-383; Kuhn, 1987, in: Structure and Function of Collagen Types (eds. Mayne, R. and Burgeson, R. E.), Academic Press, Inc., Orlando, Fla., pp. 142.
An array of conditions has been associated with the inappropriate or unregulated production of collagen, including pathological fibrosis or scarring, including endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers"" fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, cirrhosis such as binary cirrhosis and alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, scierodermaalocal, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne""s), cardiac fibrosis, muscular fibrosis/retinal separation, esophageal stricture and Pyronie""s disease. Further fibrotic disorders may be induced or initiated by surgery, including scar revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren""s contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis. Other conditions where collagen plays a key role include burns. Fibrosis of lung tissue is also observed in patients suffering from chronic obstructive airways disease (COAD) and asthma. One strategy for the treatment of these diseases and conditions is to inhibit the overproduction and/or deposition and/or unregulation of collagen. Thus, identification and isolation of molecules which control, inhibit and/or modulate the production and deposition of collagen are of major medical interest.
Recent evidence suggests that PCP is the essential key enzyme that catalyzes the cleavage of the Procollagen C-propeptide. This has been demonstrated in fibrillar collagens, including type I, type II, and type III collagen.
PCP was first observed in the culture media of human and mouse fibroblasts (Goldberg et al., 1975, Cell 4:45-50; Kessler and Goldberg, 1978, Anal. Biochem. 86:463-469), and chick tendon fibroblasts (Duskin et al., 1978, Arch. Biochem. Biophys. 185:326-332; Leung et al., 1979, J. Biol, Chem. 254:224-232). An acidic proteinase which removes the C-terminal propeptides from type I procollagen has also been identified (Davidson et al., 1979, Eur. J. Biochem. 100:551).
A partially purified protein having PCP activity was obtained from chick calvaria in 1982. Njieha et al., 1982, Biochemistry 23:757-764. In 1985, chicken PCP was isolated, purified and characterized from conditioned media of chick embryo tendons. Hojima et al., 1985, J. Biol. Chem. 260:15996-16003. Murine PCP has been subsequently purified from media of cultured mouse fibroblasts. Kessler et al., 1986, Collagen Relat. Res. 6:249-266; Kessler and Adar, 1989, Eur. J. Biochem. 186:115-121. Finally, the cDNA encoding human PCP has been identified, as set forth in the above-referenced articles and references disclosed therein.
Experiments conducted with these purified forms of chick and mouse PCP have indicated that the enzyme is instrumental in the formation of functional collagen fibers. Fertala et al., 1994, J. Biol. Chem. 269:11584.
As a consequence of the enzyme""s apparent importance to collagen production, scientists have identified a number of PCP inhibitors. See e.g., Hojima et al., supra. For example, several metal chelators have demonstrated activity as PCP inhibitors. Likewise, chymostatin and pepstatin A were found to be relatively strong inhibitors of PCP. Additionally, xcex12-Macroglobuline, ovostatin, and fetal bovine serum appear to at least partially inhibit PCP activity.
Dithiothreitol, SDS, concanavalin A, Zn2+, Cu2+, and Cd2+ are similarly reported to be inhibitory at low concentrations. Likewise, some reducing agents, several amino acids, phosphate, and ammonium sulfate were inhibitory at concentrations of 1-10 mM. Further, the enzyme was shown to be inhibited by the basic amino acids lysine and arginine (Leung et al., supra; Ryhxc3xa4nen et al., 1982, Arch. Biochem. Biophys. 215:230-235). Finally, high concentrations of NaCl or Tris-HCl buffer were found to inhibit PCP""s activity. For example, it is reported that, with 0.2, 0.3, and 0.5M NaCl, the activity of PCP was reduced 66, 38, and 25%, respectively, of that observed with the standard assay concentration of 0.1 5M. Tris-HCl buffer in a concentration of 0.2-0.5M markedly inhibited activity (Hojima et al., supra). PCP activity and its inhibition have been determined using a wide array of assays. See e.g., Kessler and Goldberg, 1978, Anal. Biochem. 86:463; Njieha et al., 1982, Biochemistry 21:757-764. As articulated in numerous publications, the enzyme is difficult to isolate by conventional biochemical means and the identity of the cDNA sequence encoding such enzyme was not known until reported in the above referenced and related patent applications.
In view of its essential role in the formation and maturation of collagen PCP appears to be an ideal target for the treatment of disorders associated with the inappropriate or unregulated production and maturation of collagen. However, none of the inhibitors so far disclosed has proven to be an effective therapeutic for the treatment of collagen-related diseases and conditions.
The identification of effective compounds which specifically inhibit the activity of PCP to regulate and modulate abnormal or inappropriate collagen production is therefore desirable and the object of this invention.
Matrix metalloproteases (MMPs) constitute a family of structurally similar zinc-containing metalloproteases, which are involved in the remodelling, repair and degradation of extracellular matrix proteins, both as part of normal physiological processes and in pathological conditions.
Another important function of certain MMPs is to activate other enzymes, including other MMPs, by cleaving the pro-domain from their protease domain. Thus, certain MMPs act to regulate the activities of other MMPs, so that over-production in one MMP may lead to excessive proteolysis of extracellular matrix by another, e.g. MMP-14 activates pro-MMP-2.
During the healing of normal and chronic wounds, MMP-1 is expressed by migrating keratinocytes at the wound edges (U. K. Saarialho-Kere, S. O. Kovacs, A. P. Pentland, J. Clin. Invest. 1993, 92, 2858-66). There is evidence which suggests MMP-1 is required for keratinocyte migration on a collagen type I matrix in vitro, and is completely inhibited by the presence of the non-selective MMP inhibitor SC44463 ((N4-hydroxy)-Nl-[(1S)-2-(4-methoxyphenyl)methyl-1-((1R)-methylamino)carbonyl)]-(2R)-2-(2-methylpropyl)butanediamide) (B. K. Pilcher, J. A. Dumin, B. D. Sudbeck, S. M. Krane, H. G. Welgus, W. C. Parks, J. Cell Biol., 1997, 137, 1-13). Keratinocyte migration in vivo is essential for effective wound healing to occur.
MMP-2 and MMP-9 appear to play important roles in wound healing during the extended remodelling phase and the onset of re-epithelialisation, respectively (M. S. Agren, Brit. J. Dermatology, 1994, 131, 63440; T. Salo, M. Makanen, M. Kylmxc3xa4niemi, Lab. Invest., 1994, 70, 176-82). The potent, non-selective MMP inhibitor BB94 ((2S,3R)-5-methyl-3{-[(1S)-1-(methylcarbamoyl)-2-phenylethyl]carbamoyl}-2-[(2-thienylthio)methyl]hexanohydroxamic acid, batimastat), inhibits endothelial cell invasion of basement membrane, thereby inhibiting anglogenesis (G. Tarboletti, A. Garofalo, D. Belotti, T. Drudis, P. Borsotti, E. Scanziani, P. D. Brown, R. Giavazzi, J. Natl. Cancer Inst., 1995, 87, 293-8). There is evidence that this process requires active MMP-2 and/or 9.
Thus PCP inhibitors which significantly inhibit MMPs 1 and/or 2 and/or 9 would be expected to impair wound healing. MMP-14 is responsible for the activation of MMP-2, and thus inhibition of MMP-14 might also result in impaired wound healing.
For recent reviews of MMPs, see Zask et al, Current Pharmaceutical Design, 1996, 2, 624-661; Beckett, Exp. Opin. Ther. Patents, 1996, 6, 1305-1315; and Beckett et al, Drug Discovery Today, vol 1(no.1), 1996, 16-26.
Alternative names for various MMPs and substrates acted on by these are shown in the table below (Zask et al, supra).
According to one aspect of the present invention, there are provided compounds of formula (I): 
wherein:
X is C1-6alkylene or C2-6alkenylene, each of which is optionally substituted by one or more fluorine atoms;
R is aryl or C3-8cycloalkyl optionally substituted by one or more fluorine atoms;
W is N or CZ;
Y and Z are each independently H, C1-4 alkyl (optionally substituted by one or more substituents independently selected from halogen, S(O)pR6, OR5, CONR1R2, CO2R7 and aryl), C1-4alkanoyl optionally substituted by one or more halogen, C1-4alkoxycarbonyl optionally substituted by one or more halogen, or CONR1R2;
R1 and R2 are each independently selected from H, C3-8cycloalkyl, C1-4alkyl (optionally substituted by C3-8cycloalkyl, aryl, CO2H, CO2R5 and/or NR3R4), or R1 and R2 can be taken together with the nitrogen to which they are attached to represent a 4- to 6-membered heterocyclic ring optionally containing one or two further hetero atoms in the ring independently selected from N, O and S, which heterocyclic ring is optionally benzo- or pyrido-fused, and which heterocyclic ring is optionally substituted by C1-4alkyl, CO2H, CO2R5, aryl and/or NR3R4;
R3 and R4 are each independently selected from H, C1-C4alkyl or C1-4alkoxycarbonyl optionally substituted by one or more halogen, or R3 and R4 can be taken together with the nitrogen atom to which they are attached to represent a morpholine, piperidine, azetidine or piperazine (optionally N-substituted by C1-4alkyl) moiety;
R5 is C1-4alkyl optionally substituted by CO2R7 or CONR3R4, or R5 is aryl;
R6 is C1-4 alkyl optionally substituted by one or more halogen, or aryl;
R7 is H or R6;
p is 0, 1 or2;
xe2x80x9cArylxe2x80x9d is a mono- or bicyclic aromatic carbocyclic or heterocyclic system comprising from 5 to 10 ring atoms, including up to 3 hetero-atoms selected from N, O and S, where, if there is a N atom in the ring, it can be present as the N-oxide, which ring system is optionally substituted by up to 3 substituents independently selected from halogen, C1-4alkyl optionally substituted by one or more halogen, C1-4 alkoxy optionally substituted by one or more halogen, phenyl, pyridyl, CO2H, CONR3R4, CO2(C1-4 alkyl), NR3R4, OH and OC(O)(C1-4alkyl);
and the pharmaceutically acceptable salts, solvates (including hydrates) and prodrugs thereof.
xe2x80x9cAlkylxe2x80x9d, xe2x80x9calkylenexe2x80x9d, xe2x80x9calkoxyxe2x80x9d, xe2x80x9calkanoylxe2x80x9d, and xe2x80x9calkenylenexe2x80x9d groups, including in groups incorporating said moieties, may be straight chain or branched where the number of carbon atoms allows.
Halogen is taken to mean fluorine, chlorine, bromine or iodine.
Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned in the art cited above, and by Berge et al, in J.Pharm.Sci., 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and indude the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, pamoate, camsylate, and p-toluenesulphonate salts.
Pharmaceutically acceptable base addition salts are well known to those skilled in the art, and for example include those mentioned in the art cited above, and can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium and zinc salts, and salts of non-toxic amines such as diethanolamine.
Certain of the compounds of formula (I) may exist in one or more zwitterionic forms. It is to be understood that pharmaceutically acceptable salts includes all such zwitterions.
Certain of the compounds of formula (I), their salts, solvates, prodrugs, etc. may exist in one or more polymorphic forms. It is to be understood that the invention includes all such polymorphs.
The compounds of formula (I), their salts, hydrates, prodrugs etc. can exhibit isotopic variation, e.g. forms with enriched 2H, 3H, 13C, 14C, 15N, 18O, etc. may be prepared, for example by suitable variation of the synthetic methods described herein using methods and reagents known in the art or routine modification thereof. All such isotopic variants are included in the scope of the invention.
Prodrug moieties are well-known to those skilled in the art (see for example the article by H Feres, in Drugs of Today, vol 19, no.9 (1983) pp.499-538, especially section A1), and for example include those specifically mentioned in A. A. Sinkula""s article in Annual Reports in Medicinal Chemistry, vol 10, chapter 31, pp.306-326, herein incorporated by reference, and the references therein. Specific prodrug moieties which may be specifically mentioned are aliphatic-aromatic, carbonate, phosphate and carboxylic esters, carbamates, peptides, glycoside, acetals and ketals, tetrahydropyranyl and silyl ethers. Such prodrug moieties can be cleaved in situ, e.g. are hydrolysable in physiological conditions, to give compounds of formula (I).
Certain of the compounds of the formula (I) may exist as geometric isomers. The compounds of the formula (I) may possess one or more asymmetric centres, apart from the specified centres in formula (I), and so exist in two or more stereoisomeric forms. The present invention includes all the individual stereoisomers and geometric isomers of the compounds of formula (I) and mixtures thereof.
Preferably the compounds of formula (I) have the following stereochemistry (IA): 
Preferably, for compounds of formula (I) where W is CZ, at least one of Y and Z is H or C1-4alkyl (optionally substituted by one or more halogen).
Preferably X is a linear C2-4alkylene moiety optionally substituted by one or more fluorine atoms. Most preferably X is propylene.
Preferably R is C3-8 cycloalkyl optionally substituted by one or more fluorine atoms.
More preferably R is cyclobutyl or cyclohexyl optionally substituted by one or more fluorine atoms. Yet more preferably R is cyclobutyl or cyclohexyl. Most preferably R is cyclohexyl.
Preferably W is N, CH or CCH3. Most preferably W is N.
Preferably Z is H or C1-4alkyl optionally substituted by one or more halogen atoms. More preferably Z is H or methyl optionally substituted by one or more fluorine atoms. Most preferably Z is H or methyl.
Preferably Y is C1-4 alkyl (optionally substituted by one or more substituents independently selected from halogen, S(O)pR6, OR5, CONR1R2, CO2R7 and aryl), C1-4alkoxycarbonyl, or CONR1R2. More preferably Y is methyl, isopropyl, methoxymethyl, 2-methoxyethyl, (pyrrolidino)COCH2, phenylsulphonylmethyl, 4-chlorophenoxymethyl, (pyridin-2-yl)methyl, (pyridin-3-yl)methyl, (pyridin-4-yl)methyl, (imidazol-2-yl)methyl, CO2(C1-2 alkyl), CONH2, CONH(C1-4alkyl (optionally substituted by C3-8cycloalkyl, aryl, CO2H or CO2R5)), CON(C1-4alkyl)(C1-4alkyl(optionally substituted by C3-8cycloalkyl, aryl, CO2H or CO2R5)), or CONR1R2 where R1 and R2 are taken together with the nitrogen to which they are attached to represent a 4- to 6-membered heterocyclic ring optionally containing one or two further hetero atoms in the ring independently selected from N, O and S, and which heterocyclic ring is optionally benzo- or pyrido-fused, and which heterocyclic ring is optionally substituted by C1-4alkyl, CO2H, CO2R5, aryl or NR3R4. Yet more preferably Y is CO2C2H5, CONH2, CONHCH3, CONH(n-C3H7), CONH(i-C3H7), (cyclopropyl)CH2NHCO, (cyclobutyl)CH2NHCO, (2-methoxyphenyl)CH2NHCO, (4-methoxyphenyl)CH2NHCO, (pyridin-2-yl)CH2NHCO, CONHCH2CO2H, CON(CH3)CH2CO2CH3, CON(CH3)2, (4-dimethylaminopiperidinyl)CO, (3-morpholinoazetidinyl)CO, (4-(pyridin-4-yl)piperidino)CO, (pyridin-2-yl)CH2N(CH3)CO, CON(CH3)CH2CO2H, (3-methoxycarbonylazetidinyl)CO, (3-carboxyazetidinyl)CO, methyl, isopropyl, methoxymethyl, 2-methoxyethyl, (pyrrolidino)COCH2, phenylsulphonylmethyl, 4-chlorophenoxymethyl, (pyridin-2-yl)methyl, (pyridin-3-yl)methyl, (pyridin-4-yl)methyl, (imidazol-2-yl)methyl, benzylaminocarbonyl, piperidinocarbonyl, (2,3-dihydroisoindol-2-yl)CO, (1,2,3,4-tetrahydroisoquinolin-2-yl)CO, morpholinocarbonyl, 4-methylpiperazinocarbonyl, (5-aza-1,2,3,4-tetrahydroisoquinolin-2-yl)CO or N-methylbenzylaminocarbonyl. Most preferably Y is CONH2, CONHCH3 or CON(CH3)2.
A preferred group of compounds is that in which each substituent is as specified in the Examples below.
Another preferred group are the compounds are those of the Examples below (especially Examples 2,3 and 12) and the salts, solvates and prodrugs thereof.
A further aspect of the invention is a PCP inhibitor which is selective against MMP-1 and/or MMP-2 and/or MMP-9 and/or MMP-14. A further aspect of the invention is the use of a PCP inhibitor which is selective against MMP-1 and/or MMP-2 and/or MMP-9 and/or MMP-14 in medicine. Further related to this aspect of the invention is the use of a PCP inhibitor which is selective against MMP-1 and/or MMP-2 and/or MMP-9 and/or MMP-14 in the manufacture of an antiscarring medicament. Further related to this aspect of the invention is a method of treating a condition mediated by PCP and in which MMP-1 and/or MMP-2 and/or MMP-9 and/or MMP-14 have a beneficial effect, with an effective amount of PCP inhibitor which is selective against MMP-1 and/or MMP-2 and/or MMP-9 and/or MMP-14, an example of such a condition being a wound. Preferably the PCP inhibitor mentioned in this aspect of the invention is selective against at least MMP-1, MMP-2 and MMP-9. Most preferably the said PCP inhibitor is selective against MMP-1, MMP-2, MMP-9, and MMP-14. Preferably the said selective PCP inhibitor has an IC50 vs. PCP of 0.5xcexcM or lower, and selectivities vs. MMP-2 and MMP-9 of at least 30-fold, in the tests described herein. Preferably the selective PCP inhibitor has an IC50 vs. PCP of 0.1 xcexcM or lower, and selectivities vs. MMP-1, MMP-2, MMP-9 and MMP-14 of at least 300-fold, in the tests described herein.
Another aspect of the invention is the use of the substances of formula (I) described herein, including the salts, solvates and prodrugs thereof, in medicine.
Another aspect of the invention is the use of the substances of formula (I) described herein, including the salts, solvates and prodrugs thereof, in the manufacture of an antiscarring medicament.
Another aspect of the invention is a pharmaceutical composition comprising a PCP inhibitor which is selective vs. MMP-1, MMP-2, MMP-9 and MMP-14, and a pharmaceutically acceptable diluent, carrier or adjuvant.
Another aspect of the invention is a pharmaceutical composition comprising a compound of formula (I), salts thereof, solvates thereof and/or prodrugs thereof, and a pharmaceutically acceptable diluent, carrier or adjuvant.
Another aspect of the invention is the combination of a PCP inhibitor, preferably a compound of formula (I), or a salt, solvate or prodrug thereof, with another material useful in treating wounds, such as:
(i) a growth factor such as TGF-xcex2-3 ( Renovo), IGF-1 (Genentech), IGF-1 complex (Celtrix), KGF-2 or FGF-10 (Sumitomo), DWP-401/EGF (Daewoong) or SNK-863 (Sanwa Kagaku Kenkyusho);
(ii) a growth factor agonist such as Noggin (Regeneron);
(iii) a growth factor antibody/antisense material, such as those to: TGF-xcex2-1 or 2 (Renovo, CaT), PDGF (II Yang) or CTGF (Fibrogen);
(iv) a hormone such as DHEAS (Pharmadigm), ConXn/Relaxin (Connetics);
(v) an antibody to adhesion compounds such as ICAM-1 (Boehringer);
(vi) a MMP such as Collagenase ABC (BioSpecifics);
(vii) a barrier such as ADCON (Gliatech);
(viii) skin products such as artificial skin systems such as those based on DermaGraft (Advanced Tissue Sciences Inc.), INTEGRA Artificial Skin (Integra Life Sciences Holding Corp.), cell cultures such as Apligraf/Graftskin (Novartis), those developed by Cell Genesys Inc., AlloDerm (LifeCell) or matrix formulation products such as Argidene gel (Telios Pharmaceuticals Inc.);
(ix) a uPA inhibitor such as those disclosed in WO 99/01451;
(x) a MMP-3 inhibitor such as those disclosed in WO 99/35124, WO 99/29667;
A further aspect of the invention is the use of a substance according to the above definitions for the manufacture of a medicament for the treatment of a condition mediated by PCP.
Yet another aspect of the invention is a method of treatment of a condition mediated by PCP comprising administration of a therapeutically-effective amount of a substance according to the above definitions.
It is to be appreciated that reference to treatment includes prophylaxis as well as the alleviation of established symptoms of PCP-mediated conditions and diseases.
The invention further provides Methods for the production of compounds of the invention, which are described below and in the Examples and Preparations. The skilled man will appreciate that the compounds of the invention could be made by methods other than those specifically described herein, by adaptation of the methods herein described in the sections below and/or adaptation thereof, for example by methods known in the art. Suitable guides to synthesis, functional group transformations, use of protecting groups, etc. are, for example, xe2x80x9cComprehensive Organic Transformationsxe2x80x9d by R C Larock, VCH Publishers Inc. (1989), xe2x80x9cAdvanced Organic Chemistryxe2x80x9d by J March, Wiley lnterscience (1985), xe2x80x9cDesigning Organic Synthesisxe2x80x9d by S Warren, Wiley Interscience (1978), xe2x80x9cOrganic Synthesisxe2x80x94The Disconnection Approachxe2x80x9d by S Warren, Wiley lnterscience (1982), xe2x80x9cGuidebook to Organic Synthesisxe2x80x9d by R K Mackie and D M Smith, Longman (1982), xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1999), and P J Kocienski, in xe2x80x9cProtecting Groupsxe2x80x9d, Georg Thieme Verlag (1994), and any updated versions of said standard works.
In the Methods below, unless otherwise specified, the substituents are as defined above with reference to the compounds of formula (I) above.
The compounds of formula (I), where W is N, can be prepared according to the chemistry outlined in the scheme below: 
The hydroxamic acid compounds of formula (I) where W is N can be made by reaction of the corresponding activated acid derivative of formula (II), where L is a suitable leaving group, with hydroxylamine.
Suitable leaving groups are generally those which would leave in a more efficient manner than the hydroxide of the parent acid (IV), in a nucleophilic substitution reaction, such as a halide, C1-4 alkoxide optionally substituted by halogen, an alkylsulphonate such as methylsulphonate or mesylsulphonate, an arylsulphonate such as p-tosylsulphonate. Other suitable leaving groups are familiar to those working in the field of amino acid coupling.
Such compounds of formula (II) may be made via standard chemistry from the corresponding acid (IV). Compounds of formula (II) where L is a leaving group such as Cl, Br, I, OCO(C1-4alkyl optionally substituted by one or more halogen), mesylate, tosylate, and the like, can be made from the corresponding compound of formula (II) where L is OH by conventional methods, including methods typified in e.g. Examples 2, etc.
The hydroxylamine used in this reaction is suitably generated in situ by treatment of a hydroxylamine salt such as the hydrochloride salt with a suitable base such as triethylamine. Suitably the reaction is carried out in a polar solvent such as DMF. This reaction, leaving groups, solvents, reagents, etc. are exemplified below in Examples 1-4, 12-16, 18, 20-28, 30, 33-40 and 41.
Alternatively the compounds of formula (I) may be made from a NHO-protected compound of formula (III), where P is a suitable O-protecting group, by suitable deprotection. Suitable O-protecting groups can be found in the text by Greene and Wuts, supra, and include trialkylsilyl (such as trimethylsilyl), benzyl, etc. Compounds of formula (III) can be made in an analogous manner to the compounds of formula (I) from the compounds of formula (II), using a protected hydroxylamine PONH2 or a suitable salt thereof in place of hydroxylamine itself or the hydroxylamine salt. The deprotection method is determined by the protective group used, as is well known in the art. E.g. benzyl groups may be hydrogenated, suitably using a catalytic transfer hydrogenation method. The reagents and conditions for reaction (III)xe2x86x92(I) are typified in Examples 29, 31, and 32 below, and also in the other Examples where a protected hydroxylamine reagent (e.g. O-trimethylsilylhydroxylamine) was used (e.g. Examples 2, etc.), where conveniently the deprotection is carried out in the same vessel as the previous step.
Other methods of making hydroxamic acids (I) are known and may be used, e.g. those mentioned in the text by J.March, supra, chapters 0-54, 0-57 and 6-4, and relevant references therein.
Acids of formula (IV) may be made by deprotection of the O-protected species of formula (V). Suitable O-protecting groups can be found in the chapter on O-protection in the book by Greene and Wuts, supra, and include C1-4alkoxy such as t-butoxy (as typified in Preparation 4), benzyloxy, trialkylsilyloxy such as trimethylsilyloxy, etc. The deprotection method is determined by the protective group used, as is well known in the art (see Greene and Wuts, supra). E.g. benzyl groups may be removed by hydrogenation, suitably using a catalytic transfer hydrogenation method, t-butyl groups may be removed by treatment with an acid such as trifluoroacetic acid, etc.
Compounds of formula (V), e.g. where P is a t-butoxy can be made for example by condensation reaction of a corresponding compound of formula (VI), for example by heating to elevated temperature in an inert solvent such as in xylene at about 130xc2x0 C., this reaction being typified by Preparation 3 below.
Compounds of formula (VI) can be made for example by coupling an acid of formula (VII) with a reagent of formula C(NH2)(Y)xe2x95x90NOH, which is available via literature methods or adaptation thereof in a conventional manner, such as typified in methods described herein (e.g. see Preparation 2, etc.). Typically the condensation is carried out by adding a solution of the acid (VII) in a suitable inert solvent such as 1,4-dioxane to a suitable agent such as 1-hydroxybenzotriazole hydrate, followed by addition of a suitable coupling agent such as a carbodiimide coupling agent, e.g. N,Nxe2x80x2-dicyclohexylcarbodiimide, then treatment with the reagent C(NH2)(Y)=NOH. Suitably the coupling is carried out at ambient temperature.
Compounds of formula (VII) can be made by hydrogenation of the corresonding itaconate derivative, which in turn can be made by conventional methods such as the Stobbe condensation. Certain aspects of these preparations related to stereoselective preparation of certain intermediates, such as are disclosed in Preparation 1xe2x80x94Route C, are novel and inventive and constitute a further aspect to this invention.
The compounds of formula (I), where W is CZ, can be prepared according to the chemistry outlined in the scheme below: 
The hydroxamic acid compounds of formula (I) where W is CZ can be made by reaction of the corresponding activated acid derivative of formula (IX), where Lxe2x88x92 is a suitable leaving group, with hydroxylamine. The leaving groups, reagents, etc. are the same as those mentioned above in relation to the corresponding compounds of formula (I) where W is N.
Such compounds of formula (IX) may be made via standard chemistry from the corresponding acdd (X) using the same or similar chemistry to that outlined above in relation to the corresponding compounds of formula (II) where W is N (supra).
Alternatively the compounds of formula (I) may be made from a NHO-protected compound of formula (VIII), where P is a suitable O-protecting group, by suitable deprotection. Suitable O-protecting groups can be found in the text by Greene and Wuts, supra, and include trialkylsilyl (such as trimethylsilyl), benzyl, etc.
Compounds of formula (VIII) can be made in an analogous manner to the compounds of formula (II) from the compounds of formula (II), using a protected hydroxylamine PONH2 or a suitable salt thereof in place of hydroxylamine itself or the hydroxylamine salt. The deprotection method is determined by the protective group used, as is well known in the art. E.g. benzyl groups may be hydrogenated, suitably using a catalytic transfer hydrogenation method. The reagents and conditions for reaction (VIII)xe2x86x92(I) are typified in Examples 29, 31, and 32 below, and also in the other Examples where a protected hydroxylamine reagent (e.g. O-trimethylsilylhydroxylamine) was used (e.g. Examples 2, etc.), where conveniently the deprotection is carried out in the same vessel as the previous step.
Other methods of making hydroxamic acids (I) are known and may be used, e.g. those mentioned in the text by J.March, supra, chapters 0-54, 0-57 and 6-4, and relevant references therein.
Acids of formula (X) may be made by deprotection of the O-protected species of formula (XI). Suitable O-protecting groups can be found in the chapter on O-protection in the book by Greene and Wuts, supra, and include C1-4alkoxy such as t-butoxy (as typified in Preparation 4), benzyloxy, trialkylsilyloxy such as trimethylsilyloxy, etc. The deprotection method is determined by the protective group used, as is well known in the art (see Greene and Wuts, supra). E.g. benzyl groups may be removed by hydrogenation, suitably using a catalytic transfer hydrogenation method, t-butyl groups may be removed by treatment with an acid such as trifluoroacetic acid, etc.
Compounds of formula (XI), e.g. where P is a t-butoxy group can be made for example by oxidation of a compound of formula (XII). Suitably the oxidation is carried out using copper (II) bromide with hexamethylenetetramine and a base such as DBU. The reagents, conditions, etc. are typified in Preparation 62 below.
Compounds of formula (XII) may be made by condensation of compounds of formula (XIII), for example by treatment of the compound of formula (XIII) with s suitable agent such as Burgess Reagent, in an anhydrous solvent such as THF. This reaction is typified in Preparation 61 below.
Compounds of formula (XIII) may be made by condensation of the acid of formula (II) above with an agent of formula NH2CH(Y)CH(Z)OH, as typified in Preparation 60 below. Compounds of formula NH2CH(Y)CH(Z)OH are available commercially, from the literature or by routine modification thereof.
Certain compounds of formula (I) may be interconverted into other compounds of formula (I)xe2x80x94for Example where Y is an acid, this can be converted to an ester and vice versaxe2x80x94typified in Examples 17 and 19 below.
It will be apparent to those skilled in the art that other protection and subsequent deprotection regimes during synthesis of a compound of the invention may be achieved by conventional techniques, for example as described in the volumes by Greene and Wuts, and Kocienski, supra.
Where desired or necessary the compound of formula (I) is converted into a pharmaceutically acceptable salt thereof. A pharmaceutically acceptable salt of a compound of formula (I) may be conveniently be prepared by mixing together solutions of a compound of formula (I) and the desired acid or base, as appropriate. The salt may be precipitated from solution and collected by filtration, or may be collected by other means such as by evaporation of the solvent.
Certain compounds of the invention may be interconverted into certain other compounds of the invention by methods mentioned in the Examples and Preparations, and well-known methods from the literature.
Compounds of the invention are available by either the methods described herein in the Methods, Examples and Preparations or suitable adaptation thereof using methods known in the art. It is to be understood that the synthetic transformation methods mentioned herein may be carried out in various different sequences in order that the desired compounds can be efficiently assembled. The skilled chemist will exercise his judgement and skill as to the most efficient sequence of reactions for synthesis of a given target compound.
The compounds, salts, solvates and prodrugs of the invention may be separated and purified by conventional methods.
Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of formula (I) or a suitable salt or derivative thereof. An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base. In certain cases preferential crystallisation of one of the enantiomers can occur from a solution of a mixture of enantiomers, thus enriching the remaining solution in the other enantiomer.
For human use, the compounds of formula (I) or their salts can be administered alone, but will generally be administered in admixture with a pharmaceutically acceptable diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they can be administered orally, including sublingually, in the form of tablets containing such excipients as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. The compound or salt could be incorporated into capsules or tablets for targetting the colon or duodenum via delayed dissolution of said capsules or tablets for a particular time following oral administration. Dissolution could be controlled by susceptibility of the formulation to bacteria found in the duodenum or colon, so that no substantial dissolution takes places before reaching the target area of the gastrointestinal tract. The compounds or salts can be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution or suspension which may contain other substances, for example, enough salt or glucose to make the solution isotonic with blood. They can be administered topically, or transdermally, in the form of sterile creams, gels, suspensions, lotions, ointments, dusting powders, sprays, foams, mousses, drug-incorporated dressings, skin patches, ointments such as petrolatum or white soft paraffin based ointments or via a skin patch or other device. They could be administered directly onto a wound. They could be incorporated into a coated suture. For example they can be incorporated into a lotion or cream consisting of an aqueous or oily emulsion of mineral oils; sorbitan monostearate; polysorbate 60; cetyl esters wax; cetearyl alcohol; 2-octyidodecanol; benzyl alcohol; water; polyethylene glycols and/or liquid paraffin, or they can be incorporated into a suitable ointment consisting of one or more of the followingxe2x80x94mineral oil; liquid petrolatum; white petrolatum; propylene glycol; polyoxyethylene polyoxypropylene compound; emulsifying wax and water, or as hydrogel with cellulose or polyacrylate derivatives or other viscosity modifiers, or as a dry powder or liquid spray or aerosol with butane/propane, HFA, CFC, CO2 or other suitable propellant, optionally also including a lubricant such as sorbitan trioleate, or as a drug-incorporated dressing either as a tulle dressing, with white soft paraffin or polyethylene glycols impregnated gauze dressings or with hydrogel, hydrocolloid, alginate or film dressings. The compound or salt could also be administered intraocularly for ophthalmic use e.g. in a lens implant or as an eye drop with appropriate buffers, viscosity modifiers (e.g. cellulose or polyacrylate derivatives), preservatives (e.g. benzalkonium chloride (BZK)) and agents to adjust tonicity (e.g. sodium chloride). Such formulation techniques are well-known in the art.
For certain uses, vaginal, rectal and nasal (e.g. by inhalation of a dry powder or aerosol) administration would be suitable.
All such formulations may also contain appropriate stabilisers and preservatives.
For oral and parenteral administration to human patients, the daily dosage level of the compounds of formula (I) or their salts will be from 0.001 to 20, preferably from 0.01 to 20, more preferably from 0.1 to 10, and most preferably from 0.5 to 5 mg/kg (in single or divided doses). Thus tablets or capsules of the compounds will contain from 0.1 to 500, preferably from 50 to 200, mg of active compound for administration singly or two or more at a time as appropriate.
For topical administration to human patients with acute/surgical wounds or scars, the daily dosage level of the compounds, in suspension or other formulation, could be from 0.01 to 50 mg/ml, preferably from 0.3 to 30 mg/ml.
The dosage will vary with the size of the wound, whether or not the wound is open or closed or partially closed, and whether or not the skin is intact.
The physician in any event will determine the actual dosage which will be most suitable for a an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
PCP Inhibition
In order to determine potency of PCP inhibitors a fluorogenic PCP cleavage assay was used. This assay is based on the template of Beekman et al. (FEBS Letters (1996), 390: 221-225) using a fluorogenic substrate. The substrate (Dabcyl-Arg-Tyr-Tyr-Arg-Ala-Asp-Asp-Ala-Asn-Val-Glu(EDANS)xe2x80x94NH2) contains the cleavage site of human PCP (Hojima et al., J Biol Chem (1985), 260: 15996-16003). Human PCP has been purified from supernatant of stable transfected CHO cells using hydrophobic interaction column followed by Superdex 200 gel filtration. 4 xcexcg total protein of this enzyme preparation was incubated with various concentrations of the substance to be tested and 3xc3x9710xe2x88x926 M substrate in assay buffer (50 mM Tris-Base, pH 7.6 containing 150 mM NaCl, 5 mM CaCl2, 1 xcexcM ZnCl2 and 0.01 % Brij 35). The assay was performed in 96-well black fluorimeter plates and fluorescence was read continuously in a fluorimeter over 2.5 hours (xcexex=340 nm, xcexem=485 nm) at a constant 37xc2x0 C. with shaking. Release of the fluorogenic signal was in linear correlation to PCP activity. Reading of the mean velocity from 30 min after start of experiment until 2.5 hours was calculated by the Biolise software. IC50 values were calculated by plotting % inhibition values against compound concentration using Tessela add in for Excel spreadsheet.
MMP Inhibition
The ability of compounds to inhibit the cleavage of fluorogenic peptides by MMPs 1, 2, 9, and 14 is described below.
The assays for MMPs 2, 9, and 14 are based upon the original protocol described by Knight et al. (Fed.Euro.Biochem.Soc., 296 (3), 263-266; 1992) with the slight modifications given below.
Inhibition of MMP-1
(i) Enzyme Preparation
Catalytic domain MMP-1 was prepared at Pfizer Central Research. A stock solution of MMP-1 (1 xcexcM) was activated by the addition of aminophenylmercuric acetate (APMA), at a final concentration of 1 mM, for 20 minutes at 37xc2x0 C. MMP-1 was then diluted in Tris-HCl assay buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 20 xcexcM ZnSO4, 0.05% Brij 35) pH 7.5 to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this assay was Dnp-Pro- -cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(N-Me-Ala)-NH2as originally described by Bickeft et al (Anal. Biochem, 212, 58-64, 1993). The final substrate concentration used in the assay was 10 xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with assay buffer so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C. prior to determination of fluorescence (substrate cleavage) using a fluorimeter (Fluostar; BMG LabTechnologies, Aylesbury, UK) at an excitation wavelength of 355 nm and emission wavelength of 440 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-2 and MMP-9
(i) Enzyme Preparation
Catalytic domain MMP-2 and MMP-9 were prepared at Pfizer Central Research. A stock solution of MMP-2/MMP-9 (1 M) was activated by the addition of aminophenylmercuric acetate (APMA). For MMP-2 and MMP-9, a final concentration of 1 mM APMA was added, followed by incubation for 1 hour at 37xc2x0 C. The enzymes were then diluted in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2 and 0.16% Brij 35, pH 7.5), to a concentration of 10 nM. The final concentration of enzyme used in the assays was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this screen was Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)NH2 (Bachem Ltd, Essex, UK) as originally described by Nagase et al (J.Biol.Chem., 269(33), 20952-20957, 1994). This substrate was selected because it has a balanced hydrolysis rate against MMPs 2 and 9 (kcat/km of 54,000, 59,400 and 55,300 sxe2x88x921 Mxe2x88x921 respectively). The final substrate concentration used in the assay was 5xcexcM.
(iii) Determination of Enzyme Inhibition
Test compounds were dissolved in dimethyl sulphoxide and diluted with test buffer solution (as above) so that no more than 1% dimethyl sulphoxide was present. Test compound and enzyme were added to each well of a 96 well plate and allowed to equilibrate for 15 minutes at 37xc2x0 C. in an orbital shaker prior to the addition of substrate. Plates were then incubated for 1 hour at 37xc2x0 C. prior to determination of fluorescence using a fluorimeter (Fluostar; BMG LabTechnologies, Aylesbury, UK) at an excitation wavelength of 328 nm and emission wavelength of 393 nm. The potency of inhibitors was measured from the amount of substrate cleavage obtained using a range of test compound concentrations, and, from the resulting dose-response curve, an IC50 value (the concentration of inhibitor required to inhibit 50% of the enzyme activity) was calculated.
Inhibition of MMP-14
(i) Enzyme Preparation
Catalytic domain MMP-14 was purchased from Prof. Tschesche, Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany. A 10 M enzyme stock solution was activated for 20 minutes at 25xc2x0 C. following the addition of 5 g/ml of trypsin (Sigma, Dorset, UK). The trypsin activity was then neutralised by the addition of 50 g/ml of soyabean trypsin inhibitor (Sigma, Dorset, UK), prior to dilution of this enzyme stock solution in Tris-HCl assay buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2 and 0.16% Brij 35, pH 7.5) to a concentration of 10 nM. The final concentration of enzyme used in the assay was 1 nM.
(ii) Substrate
The fluorogenic substrate used in this screen was Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem Ltd, Essex, UK) as described by Will et al (J.Biol.Chem., 271(29), 17119-17123, 1996). The final substrate concentration used in the assay was 10 xcexcM.
Determination of enzyme inhibition by test compounds was performed in the same manner as described for MMPs-2 and -9 above.
The compounds of Examples 1-40 and 42-58 had PCP IC50 values of 0.5 xcexcM and below, and the compounds of Examples 1-40, 43, 44 and 46 had selectivities vs MMP-2 of more than 100-fold.
All references mentioned herein in this text are incorporated by reference in their entirety.